Sperm production in the testis of human males is far less efficient than sperm production in other mammals, such as rat, rabbit and monkey (Amann, 1970) due to an increased rate of germ cell atresia. Together with this is the fact that a high incidence of sperm in the ejaculate of a fertile man is morphologically abnormal (Wyrobek et al., 1982). Thus, there is a heightened awareness of the possibility that the quantity and quality of sperm in the ejaculates of men are declining because of environmental influences (Sharpe, 1993). A toxicant-induced alteration in the process of sperm maturation during sperm transit through the epididymis, the organ in which sperm acquire fertilizing ability, could render a man infertile. It has been hypothesized that specific proteins are added to sperm in the epididymis which confer fertility. Recently, Klinefelter et al., in Journal of Andrology 15(4), 318-327 (1994) demonstrated that an 18 kD epididymal sperm surface protein, presumably a plasma membrane protein, was well correlated with fertility, although it was not believed that this protein was predictive of fertility.
There continues to be great interest in developing new and improved contraceptives. New contraceptives should be superior to existing products, e.g., oral contraceptives used by millions of women over the last 30 years are not only safe and effective, but even protect women against some cancers. However, other methods of contraception are still needed by many segments of the world's population, as many women do not have reliable access to oral contraceptives, or may suffer adverse reactions to the hormones used in oral contraceptives.
Additionally, fertility testing is becoming more widespread as increasing numbers of apparently infertile couples seek medical assistance in conception. Because reproductive abnormalities of both sexes may affect fertility, assessing male fertility is common in fertility evaluations. While the most common starting point for evaluation of male fertility is an assessment of the sperm count in semen, also important to fertility is sperm motility. Therefore, in male fertility analyses, sperm motility has also been a factor.
Currently available techniques for measuring sperm count and sperm motility are microscopic in nature. A quantitative evaluation of sperm morphology and motility requires substantial experience on the part of the laboratory technician. The high level of experience required by laboratory technicians precludes general office evaluation of semen samples and generally requires referral to a specialized laboratory. Even with adequate resources, debris in semen samples can cause erroneous or inconsistent results.
Attempts to develop biochemical assays of semen have not resulted in simple procedures which may be performed in either the physician's office or a dedicated semen evaluation lab. Most biochemical markers have failed to demonstrate correlations with sperm number, motility, or fertility. Activity of fumarase, an enzyme present in semen, has been found to correlate to both sperm count and percentage motility, Crabbe, J. Reprod. Fert. 51: 73-76 (1977). Crabbe measured fumarase activity by spectrophotometric measurements. Unfortunately, spectrophotometric assays are not generally suitable for office assays because of the cost of these specialized devices as well as the training required for accurate and reproducible operation.
Dorian, in U.S. Pat. No. 5,434,057, expanded on Crabbe's method by providing devices for assessing sperm number and motility in semen samples comprising a solid support having a carrier matrix containing a fumarase substrate and malate dehydrogenase. The sample is applied to the carrier matrix and a visual signal is detected from the solid support resulting from metabolism of the fumarase substrate by fumarase in the sample. While this assay detects motile sperm in a semen sample, there is no method for inhibiting fertility nor of selecting out the most fertile sperm in a sample.
Feuchter et al., in U.S. Pat. No. 5,250,417, disclose a method for detecting the ability of sperm to undergo the acrosome reaction to permit determination of the fertility of male mammals. The acrosome reaction is a process by which sperm release hydrolytic enzymes that degrade the zona pellucida, which must be penetrated to enable the sperm and ovum to come into contact, fuse, and complete the fertilization process.
In recent years, other studies have targeted different proteins associated with sperm in an attempt to provide new contraceptive alternatives. Major research efforts involve immunological approaches to fertility control. The development of contraceptive vaccines is directed towards the immunoneutralization of reproductive processes or interfering with fertilization by inducing antibodies against oocytes and spermatozoa. Several sperm antigens shown to have high immunocontraceptive potential are human sperm membrane antigen (SP-10) and guinea pig sperm membrane protein (PH-20). SP-10 is a sperm membrane specific antigen of 24-34 kD which was isolated using a monoclonal antibody (MHS-10) that cross-reacts with the entire acrosomal region. It is associated with the outer aspect of the inner acrosomal membrane and the inner aspect of the outer acrosomal membrane of mature human sperm. It has been reproduced recombinantly in an Escherichia coli expression system.
PH-20, a guinea pig sperm protein of 64 kD, is present on both the plasma membrane and inner acrosomal membrane of sperm. It is essential for adhesion of sperm to the zona pellucida, the initial step in the fertilization process. Active immunization with PH-20 causes infertility in both male and female guinea pigs for a period ranging from six to fifteen months.
O'Rand et al., in U.S. Pat. No. 5,175,148, disclose a sperm antigen corresponding to a sperm autoantigenic epitope which can be used as an immunocontraceptive agent as well as for diagnosing autoimmune infertility. The synthetic peptide corresponds to an autoantigenic epitope of rabbit sperm membrane autoantigen.
Several other antigens with good immunocontraceptive potential have been identified and investigated in laboratory animals, including lactic dehydrogenase-x, an isoenzyme of lactic dehydrogenase confined to male germ cells. A synthetic peptide based upon a portion of this antigen has been shown to reduce fertility in laboratory animals. Unfortunately, most studies have found that the rate and duration of the immunocontraceptive effects are less than acceptable. A problem in immunological approaches to antifertility research is the need for a safe, effective adjuvant and suitable animal models for evaluating the efficacy and safety of methods.
Although most contraceptive research has been directed to use in females, there is an interest in male fertility control both from a scientific as well as a biological viewpoint. Many compounds have been identified as having male antifertility activity in various species, e.g., gossypol, 5-thio-D-glucose, and 6-chlorodeoxyglucose. Studies have also been conducted on the use of androgens to control male fertility. Unfortunately, most compounds identified as useful in controlling male fertility appear either to have irreversible antifertility effects, to be inherently toxic, or to affect libido. It has been demonstrated that sperm count could be depressed in men injected with large doses of androgens. However, there are still questions about the potential utility of androgens as male antifertility agents. The ideal male contraceptive would produce azoospermia without compromising libido or sexual potency, and would be reversible.
While numerous sperm proteins (Primakoff et al., 1997; Burks et al., 1995; Wei et al., 1994; O'Rand et al., 1984; Lea et al., 1996; O'Rand et al., 1996; Amman et al., 1998a,b; Hammerstedt et al., 1997; Cohen et al., 1996) as well as seminal plasma proteins (Killian et al., 1993; Killian et al., 1996; Peknicova et al., 1997) have been associated with fertility over the years, in most cases they have been identified based on the demonstration of sperm-egg binding in vitro, a system that differs considerably from the natural environment of sperm.
In recent years several proteins have been linked in some way or another to fertilizing capacity of sperm. While most of these proteins are associated with the sperm membrane, some have been identified in seminal plasma. Two of the sperm proteins are quite large. The first of these is a 64 kD sperm membrane protein (PH-20) which was found to localize over the arosome of guinea pig sperm and believed to function during fertilization (Hunnicutt et al., 1996). Antibody to PH-20 has been shown to prevent binding of sperm to the zona pellucida, and males were rendered infertile when PH-20 was administered as an immune vaccine (Primakoff et al., 1997). However, PH-20 has only been described in the guinea pig, and the PH-20 vaccine severely compromises spermatogenesis in older animals, rendering the effect irreversible. In addition, a 95 kD mouse sperm protein was identified as a phosphotyrosine protein ligand for ZP3, a glycoprotein in the extracellular matrix of the egg (Burks et al., 1995). Specific peptide fragments of this protein blocked binding of sperm to the zone pellucida in vitro, but inhibition of fertility in vivo has not been demonstrated.
Several small sperm proteins have been linked to fertility. Rabbit sperm autoantigen I (RSAI), now referred to as SP-17 (Lea et al., 1996), is a 24 kD protein unique to the testis. This protein appears to be common to rabbit, mouse, and humans, as evidenced by cDNA sequence homology. Monospecific antibodies to this protein have not been used to demonstrate antifertility effects in vivo. There is no protein homology based on cDNA and deduced amino acid sequence between SP-17 and SP-22.
Protein D and E are secreted by the epithelium of the epididymis. These proteins have been linked to fertility as the p[lama membrane of the oocyte exposes protein D/E binding domains during sperm-egg fusion (Cohen et al., 1996). Antibody directed against the D/E complex significantly inhibited sperm penetration of zona-free eggs in vitro (Cuasnicu et al., 1990), but no fertility role has yet been demonstrated in vivo. While these proteins have molecular weights of 26 and 32 kD, respectively, the sequence of SP-22, the protein of the present invention, is not related to that of proteins D or E.
Wei et al, 1994, recovered a 17 kD protein from a detergent extract of human sperm. Antibody to this glycoprotein localized over the head of sperm from multiple species, but staining was localized over the enter head of the sperm as well as over the principal piece of the tail of the sperm, suggesting a lack of specificity. Thus, while the antibody inhibited fertility in vivo, much of this inhibition appears to result from nonspecific binding. Unfortunately, adequate control data were not shown. The 17 kD protein described by Wei et al. (1994) was never identified in the profile of proteins in the original detergent extract. Thus, it is impossible to determine the relationship of this protein relative to others reported in the literature. It seems that this may be similar to the more recently-described SP-17 described by Lea et al., 1996, but this is impossible to determine, as Wei et al. did not provide amino acid sequence data.
Two proteins identified in the seminal fluid, the sperm-free fraction of semen, have been linked to fertility. The first is a 17 kD protein referred to as ACR.3 (Capkova and Peknicova, 1997). This protein is a coating protein rather than an integral membrane protein and is apparently involved in mediating sperm binding to the zona pellucida, as addition of purified ACR.3 to normal sperm actually diminishes their capacity to bind to the zone. The function of these seminal plasma proteins may be to prevent premature capacitation and binding of sperm to the egg. Such membrane stabilizers may be critical to normal fertilization in vivo, but are not diagnostic of fertilizing potential. In fact, antibody to ACR.3 does not inhibit fertilization. The second seminal plasma protein that has been associated with the fertility of bull sperm is a 265 kD protein (Killian et al., 1996), now known as prostaglandin D-synthase (Gerena et all, 1998). This protein may have a more direct influence on fertility, as the addition of seminal plasma from higher fertility bulls increased the ability of sperm from lower fertility bulls to penetrate the egg in vitro. However, neither the direct addition of purified 26 kD protein, nor in vivo function tests, have been performed.